Histone H4 is a protein subunit of nucleosomes in eukaryotes and play crucial roles in DNA package and in regulation of gene expression by covalent modification. A viral histone H4 is encoded in a polydnavirus called Cotesia plutellae bracovirus (CpBV). The viral H4 (CpBV-H4) is highly homologous with other H4 proteins except 38 extended residues in N terminus. Its expression alters insect gene expression and suppresses immune and development. In this study, CpBV-H4 was expressed in a natural host, Plutella xylostella, and its suppressive activity on host gene expression was detected by suppressive subtractive hybridization (SSH) technique. SSH targets, of which expressions were down-regulated by CpBV-H4, were read by 454 pyrosequencing and annotated using the published P. xylostella whole genome. Resulting targets were assigned to most GO functional categories. Two chromatin remodeling factors were included in the SSH targets. Lysine demethylase (Px-KDM) of P. xylostella was highly expressed during entire larval period in all tested tissues. However, the suppression of Px-KDM expression by a specific RNA interference (RNAi) did not affect immune response, but significantly impaired the larval development. SWI/SNF of P. xylostella (Px-SWI/SNF) was expressed in all developmental stages. Its RNAi did not affect larval development, but led to significant alteration in adult metamorphosis. CpBV-H4 suppressed expressions of both Px-KDM and Px-SWI/SNF, but its truncated mutant lacking in the extended N-terminal tail did not. These results suggest that the developmental alteration in P. xylostella parasitized by C. plutellae can be caused by an epigenetic control of CpBV-H4 against chromatin remodeling factors.

Miniature pig sperm cryopreservation is continually researched in biotechnology for breed conservation and reproduction. It is important to control the temperature at each stage of cryopreservation and cryoprotectant. It is also necessary to find the optimal cryoprotectant concentration and chemical elements of the extender. Recently, many studies have used various cryoprotectant materials, such as dimethyl sulphoxide (DMSO), ethylene glycol (EG), antifreeze protein (AFP), amides, and glycerol. Glycerol is a commonly used cryoprotectant. However, glycerol has critical cytotoxic properties, including osmotic pressure and it can cause irreversible damage to live cells. Therefore, We focused on membrane fluidity modifications can reduce cell damage from freezing and thawing procedures and evaluated on the positive effects of trehalose to the viability, chromatin integrity, and motility of boar sperm. Miniature pig sperm was separated from semen by washing with modified- Modena B (mMB) extender. After centrifugation, the pellet was diluted with the prepared first extender. This experiment was designed to compare the effects that sperm cryopreservation using two different extenders has on sperm chromatin. The control group used the glycerol only and it was compared with the glycerol and glycerol plus trehalose extender. Sperm viability and motility were evaluated using WST1 assays and computer-assisted semen assays (CASA). Chromatin structure was examined using acridine orange staining. For the motility descriptors, trehalose caused a significant (p<0.01) increase in total motility ( in glycerol vs. in glycerol + trehalose) and progressive ( in glycerol vs. in glycerol + trehalose). A significant (p<0.05) increase in VAP ( vs. ), VSL ( vs. ), VCL ( vs. ), STR ( vs. ), and LIN ( vs. ) were also detected, respectively. The sperm DNA fragmentation index was 48.8% to glycerol only and 30.6% to glycerol plus trehalose. Trehalose added group showed higher percentages of sperm motility, stability of chromatin structure than glycerol only. In this study, we suggest that trehalose is effective in reducing freezing damage to miniature pig sperm and can reduce chromatin damage during cryopreservation.

In all the studies of mammalian species, chromatin in the germinal vesicle (GV) is initially decondensed with the nucleolus not surrounded by heterochromatin (the NSN configurations). During oocyte growth, the GV chromatin condenses into perinucleolar rings (the SN configurations) or other corresponding configurations with or without the perinucleolar rings, depending on species. During oocyte maturation, the GV chromatin is synchronized in a less condensed state before germinal vesicle breakdown (GVBD) in species that has been minutely studied. As not all the species show the SN configuration and gene transcription always stops at the late stage of oocyte growth, it is suggested that a thorough condensation of GV chromatin is essential for transcriptional repression. Because the GV chromatin status is highly correlated with oocyte competence, oocytes must end the NSN configuration before they gain the full meiotic competence and they must take on the SN or corresponding configurations to stop gene transcription before they acquire the competence for early embryonic development. In this study, we firstly investigated whether the follicle size could determine chromatin configuration in porcine oocyte. For this experiment, follicles was divided into three groups (<1 mm follicle, 1～3 mm follicle and 3～6 follicle). Using DAPI staining, the GV nucleolus and chromatin of porcine oocytes was classified into SN, SN-NSN and NSN configurations. MⅠ and M Ⅱ of three groups's Mature oocytes by staining was confirmed the configuration of chromatin. The maturation rate and parthenogenetic development potential were significant different between the SN and NSN configurations oocytes. These results indicated that chromatin changes in GV oocytes affect the development potential of porcine embryos.

In all the studies of mammalian species, chromatin in the germinal vesicle (GV) is initially decondensed with the nucleolus not surrounded by heterochromatin (the NSN configurations). During oocyte growth, the GV chromatin condenses into perinucleolar rings (the SN configurations) or other corresponding configurations with or without the perinucleolar rings, depending on species. During oocyte maturation, the GV chromatin is synchronized in a less condensed state before germinal vesicle breakdown (GVBD) in species that has been minutely studied. As not all the species show the SN configuration and gene transcription always stops at the late stage of oocyte growth, it is suggested that a thorough condensation of GV chromatin is essential for transcriptional repression. Because the GV chromatin status is highly correlated with oocyte competence, oocytes must end the NSN configuration before they gain the full meiotic competence and they must take on the SN/corresponding configurations and stop gene transcription before they acquire the competence for early embryonic development. In this study, we firstly investigated whether the layer of cumulus cells and size of oocytes could determine chromatin configurations in porcine oocytes. Using Hoechst3342 staining, the GV nucleolus and chromatin of porcine oocytes was classified into SN and NSN configurations. Next, we examined the changes in GV chromatin configurations during growth and maturation of porcine oocytes. In addition, the maturation and parthenogenetic development abilities in vitro were significant different between the SN and NSN configurations oocytes. These results indicated that chromatin changes in GV oocytes affect the development potential of parthenogenetic embryos.

The objective of this study was two folds: to investigate the relationship between paternal identification rate and sperm quality parameters such as motility and sperm chromatin structure assay after heterospermic insemination; to see if mutual complement between tests and development of useful technique to enhance the fertility in artificial insemination. In individual boar's fertilizing ability, 3 high fertility boars showed significantly high fertility (p<0.05) compared to 3 low fertility boars, but there was no difference in litter size between two groups. Sperm motility test in pooled and individual semen using computer assisted sperm analysis (CASA) revealed that no significant difference among boars. The high fertile boar showed tendency of low %Red (High red fluorescence/green+red fluorescence) in sperm chromatin structure assay (SCSA) but paternal identification rate from piglets did not differ after heterospermic insemination. The correlation coefficient between individual or pooled semen function test and farrowing rates were well correlated as follows: %Red with litter size (r= - 0.53, p=0.03); %Red with paternal identification rates (r=-0.51, p=0.03); paternal identification rates with litter size (r=0.57, p=0.02). These results indicate that sperm chromatin structure assay and sperm quality parameter test in pooled semen are useful method to predict and evaluate the fertilizing capacity after heterospermic insemination in boars.

Post-translational modifications of nucleosomal core histones play important roles in biological processes via altering chromatin structure and creating target sites for proteins acting on chromatin. Molecular genetic studies with Arabidopsis have verified several epigenetic factors that regulate flowering time. However, the roles of chromatin remodeling factors have not been well explored in rice. Here, we identified chromatin remodeling factors, OsVIL1, 2, and 4 (Oryza sativa VIN3-LIKE) genes, that regulate grain yield. OsVIL proteins contain a plant homeodomain (PHD) finger, which is a conserved motif of histone binding proteins. We showed that plant height and number of spikelets per panicle were increased in the OsVIL2-overexpression (OsVIL2-OX) and osvil4 plants, respectively. Each mutants (OsVIL2-OX and osvil4) exhibited longer internodes and thicker stems than wild type controls. Histochemical analysis revealed that cells are smaller in OsVIL2-OX and osvil4 plants. We performed an RNA-seq using 1st internodes of WT and OsVIL2-OX stems and got the suppressed target genes in the OsVIL2-OX. OsCKX2, which encodes cytokinin oxidase/dehydrogenase is one of the suppressed genes in the OX plants and we verified decrease of that gene using qRT-PCR and closed chromatins of OsCKX2 were enriched in the OX plants by using ChIP. As results of these, cytokinins were enriched in the OX plants. These demonstrate that OsVIL2 and OsVIL4 antagonistically regulate plant height and number of spikelets by controlling cytokinin contents. Like OsVIL2-OX and osvil4 plants, besides, OsVIL1-OX plants were also shown increased plant height and biomass. We propose that OsVILs may be used for improving grain yield by increasing biomass.

Flowering is exquisitely regulated by both promotive and inhibitory factors. Molecular genetic studies with Arabidopsis have verified several epigenetic repressors that regulate flowering time. However, the roles of chromatin remodeling factors in developmental processes have not been well explored in rice. We identified a chromatin remodeling factor OsVIL2 (O. sativa VIN3-LIKE 2) that promotes flowering. OsVIL2 contains a plant homeodomain (PHD) finger, which is a conserved motif of histone binding proteins. Insertion mutations in OsVIL2 caused late flowering under both long and short days. In osvil2 mutants OsLFL1 expression was increased, but that of Ehd1, Hd3a and RFT1 was reduced. We demonstrated that OsVIL2 is bound to native histone H3 in vitro. Chromatin immunoprecipitation analyses showed that OsVIL2 was directly associated with OsLFL1 chromatin. We also observed that H3K27me3 was significantly enriched by OsLFL1 chromatin in the wild type, but that this enrichment was diminished in the osvil2 mutants. These results indicated that OsVIL2 epigenetically represses OsLFL1 expression. We showed that OsVIL2 physically interacts with OsEMF2b, a component of polycomb repression complex 2. As observed from osvil2, a null mutation of OsEMF2b caused late flowering by increasing OsLFL1 expression and decreasing Ehd1 expression. Thus, we conclude that OsVIL2 functions together with PRC2 to induce flowering by repressing OsLFL1. Transgenic plants over-expressing OsVIL2 flowered early. In addition, they were taller and ticker due to increased in cell number, resulting in yield increase. The same phenotypes were observed from OsVIL4 knockout mutants. These indicate that OsVIL4 represses OsVIL2 function by directly binding to the protein.